Mutagenesis and Genotypic Characterization of Aspergillus niger FCBP-02 for Improvement in Cellulolytic Potential Shazia Shafique * , Rukhsana Bajwa and Sobiya Shafique Institute of Mycology and Plant Pathology, University of the Punjab, Quaid-e-Azam Campus, 54590-Lahore, Pakistan [email protected] Received: February 24 th , 2009; Accepted: March 18 th , 2009 Cellulase is a collective term that encompasses enzymes which catalyze reactions that participate in the degradation of insoluble cellulose to soluble carbohydrates. In the present study, production of extra cellular cellulases by a filamentous fungus, Aspergillus niger FCBP-02, was studied in solid-state fermentation (SSF) as well as in submerged fermentation (SmF). Trials were conducted to evaluate the effect of mutagenesis by UV irradiation (5–40 min) and ethyl methane sulfonate (EMS) treatment (50–300 μg mL -1 ) to obtain hyper active cellulase enzyme producers among the potential strains. The enzyme activity assays of parental and mutant strains clearly revealed significantly higher cellulase activity of mutant A-Ch-5.5 (96 Units mL -1 ), followed by A-UV-5.6 (71 Units mL -1 ) with respect to the wild strain of A. niger FCBP–02 (53.7 Units mL -1 ). The profile of genetic variability among wild and mutant derivatives was scrutinized through RAPD–PCR. The expression pattern of mutants exhibited that the mutants were isogenic variants of the wild type and the out performance of the mutants could be attributed to the change in genetic make up. Keywords: Aspergillus niger FCBP-02, cellulase activity, EMS treatment, RAPD-PCR, UV irradiation. Cellulose has attracted considerable interest as a renewable resource since bioconversion products have potential value in the food and chemical industries and as an energy source. As million of tons of cellulose are squandered annually, fungi can prove a constructive device by producing cellulases for recycling of polymeric carbohydrates, which are otherwise being locked inside the waste products [1]. The cellulolytic activities of fungi on a global scale are quite phenomenal, yet despite this, only a selected few species are cultivated to produce food products. Nowadays, several approaches including chemical mutation (nitrous acid, dimethyl sulfonate, ethylmethanesulfonate (EMS) and acridine mustards), X–rays and UV irradiation, and genetic engineering have been given a high priority to improve the yield of enzymes by fungal strains [2-5]. Such mutations are either inheritable changes or can be induced in the genome as a result of a variety of events that may involve individual nucleotide bases of the DNA molecule, sequences of bases or large regions of the fungal chromosome [6]. The present work was designed to develop mutant strains of Aspergillus niger FCBP-02 for the production of cellulases through UV and chemical mutagenesis. Research was also carried out to study the genetic variability of efficient mutant derivatives of A. niger FCBP-02 with their parental strain using RAPD markers. Strain screening: On the basis of the extent of hydrolyzing activity on solid media, strains FCBP-02 and FCBP-109 of A. niger, FCBP-64 and FCBP-209 of A. flavus, and FCBP-23 and FCBP-53 of A. fumigatus were selected to evaluate their cellulolytic activity through submerged fermentation. Maximum cellulase activity (53.7 Units mL -1 ) was recorded after 72 h of incubation with A. niger FCBP-02 (Table 1), and hence this was selected for further strain improvement. Improvement of Aspergillus niger FCBP–02 by UV mutagenesis: The ability of the mutant derivatives to utilize substrate for cellulase enzyme activity was evaluated under the preliminary growth conditions. It NPC Natural Product Communications 2009 Vol. 4 No. 4 557 - 562
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